FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

利用细胞质导入法选育优良葡萄酒嗜杀酵母

将核融合缺陷菌株嗜杀酵母5045(Kar1-1)的嗜杀质粒导入葡萄酒酵母1450(Screujsjae)中,已获成功。利用这种方法,我们选育出了具有1450细胞核和5045细胞质基因的嗜杀酵母R-58。通过萄萄酒酿造实践表明,该融合嗜杀株不仅具有抗野生酵母的能力,而且能酿制出优质葡萄酒。     

YEAST CLASSIFICATION*

Following a discussion of the need for systems of classification, which are inevitably to some extent subjective, a survey is made of primary and auxiliary diagnostic tests used in yeast classification. The possibilities and limitations of numerical taxonomy are discussed and an assessment is made of the value of current techniques used for identifying strains of brewing yeasts. Methods are described for detecting wild yeasts and for evaluating the suitability of different strains of brewing yeast. Reasons are given for retaining Saccharomyces carlsbergensis as a species in its own right rather than as a synonym of Sacch. uvarum.     

THE USE OF CONTROLLED LEVELS OF ACTIDIONE FOR BREWING AND NON-BREWING YEAST STRAIN DIFFERENTIATION

The variability of results using actidione media for the identification of beer spoilage bacteria has been shown to be due to the heat sensitivity of this reagent. Critical evaluation shows that with a particular brewery yeast, media containing 0·06 p.p.m. allow the growth of a single brewing strain but inhibit others. This permits the rapid classification of yeast strains in this pitching yeast. In a further brewery, media containing 0·16 p.p.m. differentiate brewing yeast strains from beer spoilage yeasts and this permits the quantitative assessment of these for process control at all stages of brewing. In a particular instance where a culture yeast strain is not inhibited at 0·16 p.p.m. but is p-aminobenzoic acid dependent, a differential medium incorporating this low level of actidione and omitting the vitamin allows the identification of beer spoilage yeasts in process control. These results illustrate the potential of this approach for microbiological control in specific brewery problems.     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

葡萄酒酿造过程中产β-葡萄糖苷酶酵母菌研究进展

在葡萄酒酿造的微环境中,产β-葡萄糖苷酶的酵母菌种类繁多,其酶的活性也存在着显著的差异。该文对产β-葡萄糖苷酶的酵母菌种类、产酶部位、产酶工程菌的构建、产酶酵母菌的选育方法以及酿造过程中酶解产香机制等方面的研究成果进行了综述,以期为高产β-葡萄糖苷酶酵母菌的选育和葡萄酒酿造过程中香气品质的提升提供参考。     

啤酒酵母的基因改良研究动态

近年来 ,利用基因工程进行酵母的育种在发酵广谱碳水化合物、提高糖化效率 ,改良酵母凝聚特性和改善啤酒风味方面取得了很大成绩。基因重组菌株将逐步应用到生产实践中。     

Yeasts and their importance to the flavour of traditional Chinese liquor: a review

As one of the three major distilled spirits in the world, traditional Chinese liquor has a distinctive aroma and taste. The brewing process typically involves two stages: the Daqu‐making process and the liquor‐making process. Further, it commonly adopts solid state fermentation in an open environment, which involves diverse microorganisms such as bacteria and fungi. Yeasts, as an integral brewing microorganism, are not only a dominant force in the fermentation process but also play a key role in the quality and character of different flavour liquors. Studies on yeasts associated with Chinese liquor have rarely compared them with those associated with other alcoholic beverages (wine, sake, etc.), especially in the microbiome‐related flavour of the alcoholic beverage. Here, we review the Chinese liquor brewing process, the yeast community in the brewing process, the yeast derived flavour compounds, the interaction between yeasts and other microorganisms and gene level modifications. © 2019 The Institute of Brewing & Distilling     

GROWTH OF AEROBIC WILD YEASTS

Wild yeasts of the genera Debaryomyces, Hansenula and Pichia are commonly considered to be associated with spoilage only under aerobic conditions. However, in pure cultures in either wort or a synthetic medium of yeast nitrogen base + 10% glucose, yeasts of these genera grew as well as a brewing strain of Saccharomyces cerevisiae under anaerobic conditions. Growth of S. cerevisiae was increased by the addition of unsaturated fatty acid (Tween 80) or ergosterol to the medium for anaerobic culture. No equivalent requirement was observed for the wild yeasts examined. Indeed, growth of the wild yeasts was often reduced by the addition of Tween 80, which as a surfactant prevented formation of the surface film of growth. Even under anaerobic conditions, these yeasts grew best with a surface pellicle. Although capable of good anaerobic growth in pure culture, growth of the wild yeasts was suppressed under anaerobic conditions in mixed culture with S. cerevisiae, simulating a contaminated brewery fermentation. However, the contaminants competed successfully with S. cerevisiae under aerobic conditions. There was no evidence of a “killer” effect, but prevention of pellicle formation, or production of inhibitory levels of pH or ethanol under anaerobic conditions could explain the suppression of wild yeasts under anaerobic fermentation conditions.     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Saccharomyces cerevisiae Biodiversity During the Brewing Process of an Artisanal Beer: A Preliminary Study

In this study we investigated the biodiversity of Saccharomyces cerevisiae during the brewing of an artisanal beer, as well as during its storage in the bottle for 107 days at 20°C. After inoculation with an active dried yeast (ADY), the yeast counts were followed during fermentation and after bottling. Yeast loads remained stable at 10⁶–10⁷ colony forming units (cfu)/mL, and only after day 21, were they were reduced to 10⁴ cfu/mL. After three months in the bottle they spanned 10²–10⁵ cfu/mL. Almost all isolated yeasts were identified as S. cerevisiae and after molecular characterization, unexpected results were obtained. The ADY did not take over the fermentation process and only after 21 days did isolates from the beer share similarities with the inoculated strain. During storage, a high diversity was found, underlining that each bottle developed its own micro‐ecosystem. This study highlighted the necessity for better investigations of S. cerevisiae population dynamics during artisanal brewing. Even when the chemical parameters measured confirmed a correct fermentation process, the inoculated strain was not the main yeast involved in the fermentation and consequently, the final product may have different sensory characteristics from the ones expected by the producers.     

2μm DNA PLASMID IN BREWERY YEASTS

Using an agarose gel screening procedure, 2 μm DNA plasmid was detected in all of 10 brewing strains of Saccharomyces yeast examined and in both of two non-brewing, dextrin-utilising strains. Plasmid DNA was identified in yeast grown with access to air in MYGP medium or in hopped wort, and in yeast harvested from 3-day wort fermentations. The yeast plasmid is a suitable self-cloning vector for the genetic manipulation of brewing yeasts by transformation.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

酵母菌的生存因子及其特性

麦角甾醇是酵母的“生存因子”之一,它能够延长处于衰减阶段的酵母生存时间,增强酵母菌株对酒精的抵抗能力,使酵母进一步发酵,降低葡萄酒中的残糖,但酵母细胞内麦角甾醇的富积与其生存条件、发酵环境、选择与保存条件之间有密切的关系,因而,酵母的“生存因子”对葡萄酒的酿造具有重要意义。     

RESTRICTION MAPPING OF THE POF I GENE

The gene (POFI) which imparts to certain yeasts the ability to decarboxylate phenolic acids to corresponding phenolic compounds has been analysed by restriction mapping. New restriction sites have been used to examine differences between Pof⁺ and Pof^? Saccharomyces cerevisiae strains. Southern Blot analysis of selected yeast strains has demonstrated that the POFI gene sequence is highly conserved between the Pof⁺ strain from which the gene was cloned, two Pof^? lager brewing strains and one Pof⁺ Saccharomyces brewery isolate. However, sequence differences have been found between the original Pof⁺ strain, a Pof^?laboratory strain and a Pof^? ale brewing strain.     

Recent developments in high gravity beer-brewing

Beer-brewing under high gravity (HG, specific gravity of 13–18 °P) and very high gravity (VHG, specific gravity of >18 °P) wort conditions has a wide range of benefits, from improved process economics to reduced environmental impact. This review highlights the research developments in HG and VHG brewing over the last 10 years. New research emphasizes the importance of multifaceted strategies to achieve comprehensive development in HG brewing. As the brewing yeast is exposed to a variety of stresses (e.g., osmotic stress, ethanol stress) during HG conditions, stress-alleviating strategies have been the prime focus of researchers. Studies have focused on mitigating nitrogen limitation, promoting inexpensive brewing adjuncts, and realizing the potential of exogenous enzymes in HG brewing. In addition, recent studies have addressed the importance of hybrid and mutant yeasts as well as yeast flocculation and immobilization under high specific gravity conditions. Furthermore, studies to unravel yeast physiology under HG brewing and process modelling have received much attention.     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

红曲黄酒酿造用曲及传统酿造过程中酵母菌的多样性研究

目的:探讨红曲黄酒酿造用曲中酵母菌多样性以及传统酿造过程中酵母菌菌群结构变化,为我国传统红曲黄酒中酵母菌资源的利用和对传统酿酒的有效控制及现代化酿酒新工艺的建立提供基础数据。方法:收集福建各地区的红曲黄酒酿造用曲20份,从中分离纯化出300株酵母菌,通过ITS1-5.8S-ITS2的PCR-RFLP指纹图谱对酵母菌进行分型,从各个分型类群中随机选取代表菌株,利用26S rDNA基因D1/D2区域序列分析进行分类鉴定;并采用PCR-RFLP快速分型鉴定技术分析红曲黄酒传统酿造过程中酵母菌菌群结构的变化。结果:从红曲黄酒酿造用酒曲中总共分离鉴定出12种类型酵母菌,其中扣囊复膜孢酵母(Saccharomycop-sis fibuligera)、酿酒酵母(Saccharomyces cerevisiae)和弗比恩毕赤酵母(Pichia fabianii)是酒曲中3种主要的酵母菌类型。红曲黄酒传统酿造过程酵母菌群的跟踪分析共鉴定出4种酵母菌,即酿酒酵母、扣囊复膜孢酵母、季也蒙毕赤酵母(Pichia guilliermondii)、粘性红圆酵母(Rhodotorula mucilaginosa)。在酿造前期扣囊复膜孢酵母是优势酵母菌,而在酿造的后期,酿酒酵母完全取代之成为优势菌。结论:红曲黄酒酿造用酒曲中的酵母菌具有丰富的生物多样性,红曲黄酒传统酿造过程酵母菌菌群结构处于动态变化,最终酿酒酵母成为酿造体系的优势酵母菌。